Project Summary/Abstract Intracellular pathogens drive a CD8+ T cell response typified by robust clonal expansion and phenotypic diversification into multiple subsets of effector cells. Some cells undergo terminal differentiation and are highly efficient in clearing infected cells but are short-lived. Others differentiate less, but display increased cell survival and are critical for the establishment of immunological memory. While we know that subsets of effector cells exist, the mechanisms that regulate the fate of nave CD8+ T cells after microbial challenge remain poorly understood. Previous studies suggest heterogeneity in the effector pool is driven by differences in TCR avidity, asymmetric cell division, and environmental cues. However, the developmental origin as a source of variation has been largely neglected. During immune ontogeny, the thymus is colonized by successive waves of hematopoietic stem cells (HSCs). The first major wave of HSCs is derived from the fetal liver and gives rise to the fetal and neonatal lineages of CD8+ T cells, which have an inherent propensity to rapidly proliferate and quickly become terminally differentiated after antigenic stimulation. The second wave of HSCs originates from the bone marrow and produces the adult lineages of cells, which respond less vigorously than fetal-derived CD8+ T cells but have an enhanced ability to transition into the long-lived memory pool. Using a novel fate-mapping tool, we have shown that fetal-derived CD8+ T cells persist into adulthood as a distinct developmental layer and maintain a phenotype of early response to infection and inflammatory signals. In contrast, adult-derived CD8+ T cells respond with slower kinetics but have an increased capacity to develop into memory cells. Another feature that distinguishes cells with different developmental origins is that fetal-derived CD8+ T cells are more immunodiverse than their adult counterparts, deploying a broader spectrum of effector molecules following stimulation with cognate antigen or pro-inflammatory cytokines. This data indicates that fetal-derived CD8+ T cells may make unique contributions to the CD8+ T cells response during early stages of infection. However, an important and unanswered question is how cells produced at different stages of life accomplish their division of labor. To answer this question, we will use single-cell RNAseq and dissect out how the diversity of immune functions displayed during infection is organized at the individual cell level. This is a novel and unbiased approach to deepen our conceptual understanding of the CD8+ T cell response to infection.